Method and assay kit for evaluation of the oxidative modification of protein-containing substances

ABSTRACT

Oxidative stress in living organisms is determined in a photochemiluminescence measuring system after separation of low-molecular weight antioxidants from protein-containing test samples that were withdrawn from these organisms using an assay kit that contains a gel chromatographic column, a photosensitizer solution, a carbonate buffer solution, and a phosphate buffer solution.

This is a continuation of application No. PCT/DE99/03234, filed Sep. 30,1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and assay kit for the analysisof the oxidative modification of protein-containing substances and ofthe oxidative stress in biological samples by measuring the antiradicalproperties of their protein-containing components.

2. Discussion of the Background

Oxidative stress is a common phenomenon which is implicated in theetiopathogenesis of several diseases such as atherosclerosis, cancer,acute inflammation, etc. Methods determining the concentration ofspecies reactive with thiobarbituric acid (TBA) or of conjugated dieneshave been used as routine measurements for the determination of thedegree of severity of oxidative stress. Products of lipid peroxidation,for example, malondialdehyde and 4-hydroxynonenal are reactive withthiobarbituric acid.

One drawback of the known methods for the determination of oxidativestress is the lack of specificity, because several substances react withthiobarbituric acid. Another drawback is the relative insensitivitybecause lipid peroxidation does not immediately accompany oxidativestress. In fact, lipid peroxidation occurs only after antioxidants havebeen exhausted (see FAVIER, A.: Oxidative stress: value of itsdemonstration in medical biology and problems posed by the choice of amarker, Ann. Biol. Clin. (Paris), Vol. 55, 1997, pp. 9-16).

Proteins, unlike lipids, react immediately to oxidative stress.Different alterations, particularly in amino acids are detected inprotein degradation assays (see PACIFICI, Robert E.; DAVIES, Kelvin J.A.: Protein Degradation as an Index of Oxidative Stress. In: METHODS INENZYMOLOGY, Vol. 186, Part B, Eds. Packer, L. and Glazer, A. N. AcademicPress, Inc. 1990, pp. 485-502). These alterations include formation ofcharacteristic products, alterations in the secondary, tertiary andquaternary structure, electric charge, folding, hydrophobicity,fragmentation, covalent inter- and intramolecular cross-linkage orincrease in proteolytic sensitivity. However, determination of theseparameters is very complicated, expensive, cumbersome and oftennon-specific, requiring methods such as radioactive or fluorescentlabeling, gel electrophoresis, Western blots and immunoprecipitation.

Accordingly, there has been a need for a simplified method that allowsdetermination of oxidative stress in organisms and the evaluation ofantiradical activity of substances, particularly without theinterference of low-molecular weight antioxidants, such as ascorbic acidand uric acid.

SUMMARY OF THE INVENTION

It is an objective of the present invention to devise a new method forthe determination of oxidative stress in an organism. Another objectiveis to devise an assay kit for the measurement of oxidative stress in anorganism by investigation of body fluids, for example blood plasma.

These and other objects are achieved according to the invention, thefirst embodiment of which includes a method for quantitative analysis ofthe oxidative modification of a protein-containing substance,comprising:

purifying said protein-containing substance, thereby removing alow-molecular weight antioxidant and providing a purifiedprotein-containing substance;

generating free radicals in said purified protein-containing substance;

measuring the antiradical properties of said purified protein-containingsubstance in a free-radical generating measuring system.

Another embodiment of the invention includes an assay kit for theanalysis of oxidative modification of protein-containing substances,comprising:

a gel chromatographic column, an aqueous photosensitizer solution and anaqueous carbonate buffer solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

Antioxidant capacity (ACW) of methionine, measured in a 2 mmol/lsolution in H₂O (lower curve) and human serum albumin (HSA) in a 60 g/lsolution in H₂O (upper curve) during irradiation with UV light (λ=254nm) in equivalent concentration of ascorbic acid (calibrationsubstance).

FIG. 2:

Antioxidant capacity of histidine (2 mmol/l) under conditions ofchemical (NaOCl) and physical (UV, λ=254 nm) oxidation in equivalentconcentration of ascorbic acid (calibration substance).

For UV: dose 1=60 sec, dose 2=120 sec.

For NaOCl: after 45-minute incubation with 16 (dose 1) or 32 (dose 2)mg/l NaOCl.

FIG. 3:

Antioxidant capacity of LDL in equivalent concentration of Trolox®(calibration substance) during Cu²⁺-induced oxidation.

FIG. 4:

Results of antioxidant capacity of plasma protein (ACP) measurements inhealthy donors and cancer patients, 1 ASA=10 pmol/Asc/mg protein.

Mean values: 13.5 and 21.6 ascorbic acid equivalents (ASA); errorprobability p<0.0005.

DETAILED DESCRIPTION OF THE INVENTION

Free radicals are associated with oxidative stress and can be generatedby a variety of methods, i.e., physical (radiolysis, photolysis,electrolysis, etc.), physico-chemical (thermal decomposition of nitrogencompounds, photosensitized generation), chemical (Fe⁺⁺/H₂O₂ system, KO₂decomposition, autoxidation of several compounds), and biochemical fromindividual enzymes (e.g., xanthine oxidase) to subcellular fractions(NADPH-consuming microsomes). The effect of the antioxidants can bedetected by measurement of O₂ consumption, light absorption, electricalconductivity, fluorescence, and chemiluminescence.

While investigating the mechanism of the therapeutic efficacy ofultraviolet irradiation of blood, it was surprisingly found that theantioxidant capacity of blood plasma increases during UV-B irradiation,rather than, as expected, declines. An in-depth investigation of thisphenomenon has revealed that this increase in antioxidant capacity canbe attributed to seven amino acids (cysteine, histidine, methionine,phenylalanine, serine, tryptophan and tyrosine). Their antioxidantcapacity is unfolded during irradiation and increases in accordance withthe dose. The antioxidant capacity of human serum albumin (HSA)undergoes similar changes during oxidation. As shown in FIG. 1, theantioxidant capacity (ACW) of human serum albumin increases during thecourse of irradiation with UV-light (λ=254 nm). An increase ofantioxidant capacity has also been found during irradiation of histidinesolutions in which oxidative stress was induced by NaOCl or UV-light(λ=254 nm) (FIG. 2). The effects of Cu²⁺-induced oxidation of LDL (lowdensity lipoprotein) on its antioxidant capacity are shown in FIG. 3.

These so far unknown properties of blood plasma provide the basis forthe determination of oxidative stress in living organisms byinvestigation of their protein-containing components.

The method according to the invention comprises the following steps:

A test sample is obtained from an organism, for example blood plasma orany other protein-containing substance. For example, blood is withdrawnfrom the cubital vein of a human. The blood plasma can be separated fromthe cells by, for example, centrifugation. The test sample, such asblood plasma, is then passed through a gel-chromatographic column. Suchcolumn can be a desalting column, for example, an Econo-Pac™ 10DG columnfrom Bio-Rad which contains Bio-Gel P-6 gel. The desalted test sample iseluted from the column with a phosphate buffer saline (PBS). Thephosphate buffer saline can be prepared, for example, by adding 8.18 gNaCl and 3.58 g Na₂HPO₄x12H₂O to 1 L H₂O, and by adjusting the pH withHCl to 7.4.

The assay kit according to the present invention for the determinationof oxidative stress in an organism by photochemiluminescence (PCL)investigations comprises a buffer and a photosensitizer. Preferably, thebuffer has a basic pH value. More preferably the pH is between 7-14.Most preferably the pH is 10.6. The pH value includes all valuestherebetween, and especially including 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,10.5, 11, 11.5, 12, 12.5, 13, and 13.5. Alkali metal carbonates andearth alkali metal carbonates are preferably used as carbonates. Morepreferably, sodium carbonate is used as carbonate. The bufferconcentrate can have a molarity of 0.1-0.3. Preferably, a 0.2 molarbuffer solution in H₂O is used. Different types of photosensitizer canbe used, for example, riboflavine and methylene blue. Preferably luminolis used as a photosensitizer.

The eluate from the desalting column is added to the solution comprisingthe buffer and the photosensitizer. The solution can also containdeionized water.

The antioxidant capacity is preferably measured in aphotochemiluminescence (PCL) measuring system. Free radicals can begenerated in a free-radical generating system such as aphotochemiluminescence (PCL) measuring system. Such system can be aPhotochem® unit consisting of a cell for irradiation, a low pressuremercury lamp, a peristaltic mini-pump, a chemiluminescence measuringcell, and a personal computer. (Popov, I., Lewin, G. Antioxidativehomeostasis: characterization by means of chemiluminescent technique.In: METHODS IN ENZYMOLOGY, Vol. 300, Eds. Packer, L. and Glazer, A. N.,Academic Press, New York, 1999, p.p. 437-456).

Surprisingly, it was revealed that the antiradical (antioxidant)capacity of the proteins obtained from blood plasma increases in adose-related manner after oxidatiye stress caused by both chemical (e.g.hypochlorite) and physical (e.g. ultraviolet light) factors. Thiscapacity remains unchanged for at least 24 hours after treatment.Accordingly, it is possible to quantitatively determine the degree ofoxidative stress in the organism after withdrawing blood and separationof proteins.

The oxidative capacity of a substance can be evaluated according to thedegree of change of a parameter of the registered curve. This is theso-called lag-phase. the longer the lag-phase, the higher theantioxidative capacity of the substance. The antioxidative capacity ofdifferent substances is compared to a standard substance (calibrationsubstant). Preferably ascorbic acid is used as standard substance.Accordingly, the antioxidative capacity is expressed in concentrationunits (mmol/l) of ascorbic acid, that has the same activity (lag-phase)in the measuring system.

The calibration proceeds as follows:

Solutions of different concentrations of ascorbic acid are prepared,containing, for example, 1, 2, 3, 4, and 5 nmol of ascorbic acid. Adiagram for the dependence of the lag-phase from the concentration ofthe acid is then prepared based on the measurement of the antioxidativecapacity of ascorbic acid. The lag-phase of measured samples can theneasily be correlated to a specific concentration of the ascorbic acid.Accordingly, the measuring results can be evaluated in the PCL measuringsystem in equivalent concentrations of a suitable calibration substance(ascorbic acid or Trolox® consisting of a water soluble derivative ofalpha-tocopherol), but also in absolute terms in seconds of the lagphase, of the point of inflection (maximum value of the firstderivative) or as a percentage of inhibition (with the integral asevaluation parameter) of the PCL curves.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1

0.5 ml blood plasma were eluted with a phosphate buffer solution in adesalting column manufactured by BIO-Rad. After passage of 2.5 ml of theeluent, 15 μl of the eluate from the subsequent total volume of 2 mlwere measured in the PCL measuring system. The displacement of the pointof inflection of the recorded curve, compared with that of thecorresponding calibration curve, was used for conversion to theequivalent effectiveness of ascorbic acid. The resulting antioxidantcapacity of the plasma proteins (ACP) was calculated in nmol ascorbicacid per mg protein, while taking account of the protein concentrationin the sample (determined e.g. with the biuret method), and illustratedin ascorbic acid equivalents (ASA).

Definition: 1 ASA=10 pmol Asc/mg protein.

Example 2

The assay kit for 100 measurements consisted of 4 desalting columns and6 bottles:

1 bottle with 120 ml 0.2 M sodium carbonate solution with 20 mg EDTA.

1 bottle with 200 ml deionized water.

4 black bottles with 0.75 ml luminol solution (3 mmol/l) (portions anddark coloring are required due to pronounced light sensitivity).

The entire measurement operation was performed as follows:

1 ml buffer was mixed with (1.5-x) ml water and x μl of a proteinsolution (for blank value, pure PBS solution) is added. x is 15 μl forthe eluate. Then 25 μl of the luminol solution were injected. Themixture was transferred to the photochemiluminometer and the measurementwas started.

After the measurement, the instrument was rinsed twice with deionizedwater.

Example 3

Human blood samples were examined according to the measuring principledescribed above. Blood was withdrawn from the cubital vein, using EDTAas an anticoagulant. Immediately after withdrawal, the plasma wasseparated from cells by means of centrifugation and frozen until thetime of measurement.

After the measurement according to Example 2 the results were evaluatedon the basis of displacement of the point of inflection. Twenty healthydonors and 18 patients with breast cancer were examined. The results areshown in FIG. 4. The mean value for antioxidant capacity of plasmaproteins ACP in healthy donors was 13.5 ASA and 21.6 ASA in cancerpatients. The significance of the higher oxidative stress in cancerpatients known from the literature was estimated according to thisparameter with the error probability p≦0.0005. The error probability wasdetermined using the T-test of an Excel-97 program. References citingdata for oxidative stress in cancer include: Clin. Biochem., March 1999,32(2):131-6; Free Radic. Biol. Med., February 1999; 26(3-4):410-8; J.Am. Diet. Assoc., May 1998; 98(5):524-8; Chem. Res. Toxicol., December1996; 9(8):1285-92; Cancer Epidemiol. Biomarkers Prev., September 1996;5(9):705-10; Carcinogenesis, November 1994; 15(11):2637-43; Eur. J.Clin. Nutr., August 1994; 48(8):575-86; J. Cancer Res. Clin. Oncol.1994; 120(6):374-7; Int. J. Epidemiol. August 1992; 21(4):625-35; Clin.Biochem., July 1999; 32(5):369-73; Biochem. Int., September 1991;25(2):371-80; and J. Nutr., April 1996; 126(4 Suppl):1201S-7S.

The priority document of the present application, German patentapplication, DE 198 46 148.8, filed Oct. 1, 1998 and PCT application,PCT/DE99/03234, filed Sep. 30, 1999, are incorporated herein byreference.

Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed is:
 1. An assay kit for the analysis of oxidativemodification of protein-containing substances, consisting essentiallyof: a gel chromatographic column; an aqueous photosensitizer solution;an aqueous carbonate buffer solution; optionally water; and optionallyan aqueous phosphate buffer solution.
 2. The assay kit according toclaim 1, wherein said photosensitizer solution sensitizes an oxidativelymodified protein-containing substance.
 3. The assay kit according toclaim 1, wherein said gel chromatographic column is a desalting column.4. The assay kit according to claim 1, wherein said aqueousphotosensitizer solution is a luminol solution.
 5. The assay kitaccording to claim 1, wherein said aqueous photosensitizer has aconcentration of 3 mmol/l.
 6. The assay kit according to claim 1,wherein said aqueous carbonate buffer solution contains sodiumcarbonate.
 7. The assay kit according to claim 1, wherein said aqueouscarbonate buffer solution has a concentration of 0.2 mol/l.
 8. The assaykit according to claim 1, wherein said carbonate buffer has a pH of10.6.
 9. The assay kit according to claim 1, wherein said phosphatebuffer solution is obtained by adding 8.18 g NaCl, 3.58 g Na₂HPO₄x12H₂Oto 1 L H₂O, and by adjusting the pH with hydrochloric acid.
 10. Theassay kit according to claim 9, wherein said pH is 7.4.
 11. An assay kitfor the analysis of oxidative modification of protein-containingsubstances, consisting essentially of: a gel chromatographic column; anaqueous photosensitizer solution; an aqueous carbonate buffer solution;an aqueous phosphate buffer solution; and optionally water.
 12. An assaykit for the analysis of oxidative modification of protein-containingsubstances, consisting essentially of: a gel chromatographic column; anaqueous photosensitizer solution; an aqueous carbonate buffer solution;water; and optionally an aqueous phosphate buffer solution.